EP1091973A4 - HYBRIDATION $i(IN SITU) SOUS FLUORESCENCE A PARAMETRES MULTIPLES - Google Patents

HYBRIDATION $i(IN SITU) SOUS FLUORESCENCE A PARAMETRES MULTIPLES

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Publication number
EP1091973A4
EP1091973A4 EP99955269A EP99955269A EP1091973A4 EP 1091973 A4 EP1091973 A4 EP 1091973A4 EP 99955269 A EP99955269 A EP 99955269A EP 99955269 A EP99955269 A EP 99955269A EP 1091973 A4 EP1091973 A4 EP 1091973A4
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European Patent Office
Prior art keywords
probes
chromosome
labeled
fluorophores
label
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EP99955269A
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German (de)
English (en)
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EP1091973A1 (fr
Inventor
David C Ward
Michael Speicher
Stephen Gwyn Ballard
John T Wilson
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Yale University
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Yale University
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Priority claimed from US09/088,845 external-priority patent/US6007994A/en
Application filed by Yale University filed Critical Yale University
Publication of EP1091973A1 publication Critical patent/EP1091973A1/fr
Publication of EP1091973A4 publication Critical patent/EP1091973A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to nu ⁇ _Ieic acid chemisiiy, and more specifically to reagents and methods for accomplishing multiplex image analysis of chromosomes and chromosomal fragments.
  • the invention may be used to diagnose chromosomal abnormalities, infectious agents, etc. This invention was made in part using
  • chromosome identification has involved the use of labeled chromosome-specific oligonucleotide probes to label repetitive sequences of interphase chromosomes (Cremer, T. et al., Hum. Genet. 74:346-352 (1986); Cremer, T. et al., Exper. Cell Res. 176:119-220 (1988)).
  • Such methods have been shown to be useful in the prenatal diagnosis of Down's Syndrome, as well as in the detection of chromosomal abnormalities associated with tumor cell lines.
  • Chromosome-specific probes of repetitive DNA that localize to discrete sub- regions of a chromosome are, however, unsuitable for analyses of many types of chromosomal abnormalities (e.g., translocations or deletions).
  • Ward, D.C. et al. discloses a chromosomal in situ suppression ("OSS") hybridization method for specifically labeling selected mammalian chromosomes in a manner that permits the recognition of chromosomal aberrations.
  • OSS chromosomal in situ suppression
  • sample DNA ii denatured and permitted to hybridize with a mixture of fluorescently labeled chromosome-specific probes having high genetic complexity and unlabeled nonspecific competitor probes.
  • Chromosomal images were obtained as described by Manuelidis, L. et al. (Chromosoma 95:397-410 (1988), herein incorporated by reference).
  • the method provides a rapid and highly specific assessment of individual mammalian chromosomes.
  • the method permits, by judicious selection of appropriate probes and/or labels, the visualization of sub-regions of some or all of the chromosomes in a preparation. For example, by using more than one probe, each specific for a sub-region of a target chromosome, the method permits the simultaneous analysis of several sub-regions on that chromosome.
  • the number of available fluorophores limits the number of chromosomes or chromosomal sub-regions that can be simultaneously visualized.
  • a "combinatorial" variation of the CISS method can be employed.
  • two fluors permit three different chromosomes or chromosomal sub-regions to be simultaneously visualized.
  • a hybridization probe mixture is made from a single set of probe sequences composed of two halves, each separately labeled with a different fluorophore.
  • the two fluorophores Upon hybridization, the two fluorophores produce a third fluorescence signal that is optically distinguishable from the color of the individual fluorophores.
  • Extension of this approach to Boolean combinations of n fluorophores permits the labeling of 2 n - ⁇ chromosomes.
  • the invention concerns reagents and methods for combinatorial labeling of nucleic acid probes sufficient to permit the visualization and simultaneous identification of all 22 autosomal human chromosomes and the human X and Y chromosomes, or defined sub-regions thereof. Such specific labeling of entire chromosomes or defined sub-regions thereof is referred to as "painting.”
  • the invention further concerns reagents and methods for combinatorial labeling of nucleic acid probes sufficient to permit the characterization of bacteria, viruses and/or lower eukaryotes that may be present in a clinical or non-clinical preparation.
  • the invention concerns a set of combinatorially labeled oligonucleotide probes comprised of a first and a second subset of probes, wherein:
  • each member of the first subset of probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subsets of probes, and (ii) being capable of specifically hybridizing with one predetermined autosomal or sex chromosome of a human karyotype; the first subset of probes set having sufficient members to be capable of specifically hybridizing each autosomal or sex chromosome of the human karyotype to at least one member; and
  • each member of the second subset of probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subset, and (ii) being capable of specifically hybridizing with one extra-chromosomal polynucleotide copy of a predetermined region of an autosomal or sex chromosome of the human karyotype.
  • the invention further concerns a set of combinatorially labeled oligonucleotide probes comprised of a first subset of genotypic probes and a second subset of phenotypic probes, wherein:
  • each member of the first subset of genotypic probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subsets of probes, and (ii) being capable of specifically hybridizing with a region of a nucleic acid of a preselected bacterium, virus or lower eukaryote; the first subset of probes set having sufficient members to be capable of distinguishing the preselected bacterium, virus, or lower eukaryote from other bacteria, viruses, or lower eukaryotes; and
  • each member of the second subset of phenotypic probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subset, and (ii) being capable of specifically hybridizing with a predetermined polynucleotide region of the chromosome of the preselected bacterium, virus, or lower eukaryote, or an extra-chromosomal copy thereof so as to permit the determination of whether the preselected bacterium, virus, or lower eukaryote exhibits a preselected phenotype.
  • the invention additionally concerns a method of simultaneously identifying and distinguishing the individual autosomal and sex chromosomes of a human karyotype which comprises the steps:
  • each member of the first subset of probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subsets of probes, and (ii) being capable of specifically hybridizing with one predetermined autosomal or sex chromosome of a human karyotype; the first subset of probes set having sufficient members to be capable of specifically hybridizing each autosomal or sex chromosome of the human karyotype to at least one member; and (B) each member of the second subset of probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subset, and (ii) being capable of specifically hybridizing with an a predetermined extra- chromosomal polynucleotide copy of a region of an autosomal or sex chromosome of the
  • LT for each chromosome of the preparation hybridized to a member of the first subset of probes, detecting and identifying the predetermined label of that member and correlating the identity of the label of that member with the identity of the autosomal or sex chromosome of the human karyotype with which that member specifically hybridizes, to thereby identify the chromosome hybridized to the member;
  • (LTI) repeating step (LT) until each autosomal and sex chromosome of the human karyotype has been identified in the preparation (IV) for each member of the second subset of probes hybridized to a predetermined extra-chromosomal polynucleotide copy of a region of an autosomal or sex chromosome detecting and identifying the predetermined label of that member and correlating the identity of the label of that member with the identity of the region of the autosomal or sex chromosome of the human karyotype with which that member specifically hybridizes, to thereby identify the region of
  • the invention additionally concerns a method of simultaneously identifying and distinguishing a preselected bacterium, virus, or lower eukaryote from other bacteria, viruses or lower eukaryotes that may be present in a sample which comprises the steps: (I) contacting a preparation suspected to contain the preselected bacterium, virus, or lower eukaryote, under conditions sufficient to permit in situ nucleic acid hybridization to occur, with a set of combinatorially labeled oligonucleotide probes comprised of a first subset of genotypic probes and a second subset of phenotypic probes, wherein: (A) each member of the first subset of genotypic probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subsets of probes, and (ii) being capable of specifically hybridizing with a region of a nucleic acid of the preselected bacterium
  • step (V) repeating step (IV) for each member of the second subset of probes.
  • the invention particularly contemplates the embodiments in which the members of the above sets of probes are detectably labeled with fluorophores, and, wherein at least one member of the set is combinatorially labeled with either one, two, three, four or five fluorophores selected from the group consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each member of the set is labeled with at least one fluorophore selected from the fluorophore group.
  • the invention additionally provides a set of combinatorially labeled oligonucleotide probes, each member thereof: (i) having a predetermined label distinguishable from the label of any other member of the set, and (ii) being capable of specifically hybridizing with a telomeric region of one predetermined autosomal or sex chromosome of a human karyotype; the set having sufficient members to be capable of specifically hybridizing each autosomal or sex chromosome of the human karyotype to at least one member.
  • the invention additionally provides a method of simultaneously identifying and distinguishing the individual autosomal and sex chromosomes of a human karyotype which comprises the steps:
  • step (c) repeating step (b) until each autosomal and sex chromosome of the human karyotype has been identified in the preparation.
  • Figure 1 provides a schematic illustration of a CCD camera and microscope employed in accordance with the present methods.
  • Figure 2 shows the raw data from a karyotypic analysis of chromosomes from a bone marrow patient (BM2486). Adjacent to each source image is a chromosome "mask" generated by the software program.
  • panels A and B are the DAPI image and mask
  • panels C and D are FITC image and mask
  • panels E and F are Cy3 image and mask
  • panels G and H are Cy3.5 image and mask
  • panels I and J are Cy5 image and mask
  • panels K and L are Cy7 image and mask.
  • Figures 3A and 3B show the identification of individual chromosomes by spectral signature of patient BM2486.
  • Figure 2 is the same photograph as Figure 3A, except that it is gray scale pseudocolored.
  • Figure 3B displays the karyotypic array of the chromosomes.
  • Figure 4 shows the differentiation of bacteria by in situ hybridization.
  • Panels A-E represent in situ hybridization assay results on a laboratory derived mixture of three bacteria (F. nucleatum, A. actinomycetemcomitans and E. corrodens) using a mixture of genomic DNA probes for each organism.
  • Panel A shows all the bacteria present in the microscope field detected by (DAPI), a general DNA binding fluorophore.
  • Panel B shows F. nucleatum using Cascade Blue detection.
  • Panel C shows A. actinomycetemcomitans using FTTC detection.
  • Panel D shows E. corrodens using Rhodamine detection.
  • Panel ⁇ is a computer-merged composite of panels B-D showing the differentiation of each bacteria in the sample.
  • Panel F is a similar analysis of a mixture of C. gingivalis (using rhodamine detection) and P. intermedia (using FTTC detection) when hybridized with a combination of genomic DNA probes for those organisms.
  • Panel G shows an in situ hybridization assay for a combination of seven different bacteria hybridized with a mixture of seven genomic DNA probes. In panel G, the numbers (l)-(7) identify the bacteria.
  • F. nucleatum (1), E. corrodens (2) and A. actinomycetemcomitans (3) are shown in blue (Cascade Blue detection).
  • P. gingivalis (4) and C. ochracea (5) are shown in red (Rhodamine detection), whereas P. intermedia (6) and C.
  • Panel H represents an in situ hybridization assay for the presence of A. actinomycetemcomitans (using FITC detection) in a plaque sample obtained from a patient with localized juvenile periodontist.
  • Figure 5A shows a normal male metaphase chromosomal spread after hybridization with a 24-color set of telomere-specific probes, shown as a pseudocolorized image.
  • Figure 5B shows the final karyotype generated on the basis of the boolean spectral signature of the telomere-specific probes.
  • Figure 6A shows the hybridization pattern of the chromosome 8 subtelomeric YACs (telomere-specific probes) on a normal metaphase chromosomal pread.
  • Figure 6B shows the hybridization pattern of the same probes (as those used in Figure 6A) on a metaphase chromosomal spread from a patient with a myeloproliferative disorder. Previous cytogenetic analysis of this patient using G-banding revealed a trisomy 8 as the only change; the M-FISH telomere-specific probes show a split telomere signal on the short arms of two of the three chromosome 8, indicating an additional change: an inversion in this re "gtri*on.
  • Fluorescence in situ hybridization is used in a variety of areas of research and clinical diagnostics (Gray, J.W. et al, Curr Opin Biotech 5:623-631 (1992); Xing, Y. et al., In: The Causes and Consequences of Chromosomal Aberrations. I.R. Kirsch Ed. CRC Press, Boca Raton, pages 3-28 (1993)).
  • FISH Fluorescence in situ hybridization
  • FISH FISH offers the capacity for multiparameter discrimination. This allows the simultaneous visualization of several DNA probes using either a combinatorial (Nederiof, P.M. et al., Cytometry 10:20-21 ( 1989); Nederiof, P.M. et al., Cytometry 77: 126-131 (1990); Ried, T. et al., Proc Natl Acad Sci (U.S.A.) 89: 1388-1392 (1992a); Ried, T. et al., Hum Mol Genet 7:307-313 (1992b); Lengauer, C. et al., Hum Mol Genet 2:505-512 (1993); Popp, S.
  • the invention concerns a set of combinatorially labeled oligonucleotide probes, each member thereof: (i) having a predetermined label distinguishable from the label of any other member of the set. and (ii) being capable of specifically hybridizing with one predetermined autosomal or sex chromosome of a human karyotype.
  • the set will have a sufficient number of members to be capable of specifically and distinguishably hybridizing each autosomal or sex chromosome of said human karyotype to at least one member.
  • the term "karyotype" denotes the compliment of chromosomes found in a normal or aberrant cell.
  • the number of chromosomes is 46, comprising 22 pairs of autosomal chromosomes and 2 sex chromosomes (either 2 X chromosomes (if female) or an X and Y chromosome (if male)).
  • the labels are said to be distinguishable in that the particular label of any one member of the set (and the identity of that member) differ from the particular label and identity of any other member of the set.
  • each probe member is capable of specifically hybridizing to only one chromosome (or sub- chromosomal region) and since the identity of the label and probe are known in advance, the detection of a particular label associated with an unidentified chromosomal region means that the probe bearing that label has become hybridized to the unidentified chromosomal region. Since the chromosome to which that probe specifically hybridizes is known, the detection of a distinguishable label permits the identification of the chromosomal region. More specifically, the invention concerns fluors that can be used to label oligonucleotide probes so that such probes may be used in multiparametric fluorescence in situ hybridization.
  • a "fluor” or “fluorophore” is a reagent capable of emitting a detectable fluorescent signal upon excitation. Most preferably, the fluor is coupled directly to the pyrimidine or purine ring of the nucleotides of the probe (Ried, T. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 89: 1388-1392 (1992), herein incorporated by reference; U.S. Patent Nos. 4,687,732; 4,711,955; 5,328,824; and 5,449,767, each herein incorporated by reference.
  • the fluor may be indirectly coupled to the nucleotide, as for example, by conjugating the fluor to a ligand capable of binding to a modified nucleotide residue.
  • ligands capable of binding to a modified nucleotide residue.
  • the most preferred ligands for this purpose are avidin, streptavidin, biotin-binding antibodies and digoxigenin-binding antibodies. Methods for performing such conjugation are described by Pinkel, D. et al, Proc. Nat'l. Acad. Sci. (U.S.A.) 85:2934-2938 (1986), herein incorporated by reference).
  • multiparametric fluorescence denotes the combinatorial use of multiple fluors to simultaneously label the same chromosome or sub-chromosomal fragment, and their detection and characterization. Chromosomes or sub- chromosomal fragments are said to be simultaneously labeled if they are exposed to more than a single chromosome-specific probe under conditions sufficient to permit each chromosome-specific probe to independently hybridize to its target chromosome . As used herein, it is thus unnecessary for all such hybridization reactions to commence and conclude at the same instant. The simultaneous labeling permitted by the present invention is thus in contrast to protocols in which chromosomes are exposed to only a single chromosome-specific probe at a time.
  • the simultaneous detection and characterization permitted by the present invention denotes an ability to detect multiple (and most preferably all) of the autosomal and/or sex chromosomes in a sample, without any need to add further reagent, or probe after the detection of the first chromosome.
  • digital images of the chromosomes are obtained for each fluorophore employed, thereby providing a series of gray scale fluorescence intensities associated with each fluorophore and each chromosome.
  • the final image is obtained by pseudocoloring the blended gray scale intensities for each chromosome.
  • the invention thus provides a method of simultaneously identifying and distinguishing the individual autosomal and sex chromosomes of a human karyotype which comprises contacting a preparation of chromosomes, that has been previously treated to render it in single-stranded form, with the above-described set of combinatorially labeled oligonucleotide probes, under conditions sufficient to permit nucleic acid hybridization to occur.
  • Such treatment causes at least one of each autosomal or sex chromosome of the preparation to become hybridized to at least one member of said set of probes.
  • oligonucleotide probes used in accordance with the methods of the present invention are of either of two general characteristics.
  • such probes are chromosome or sub-chromosome specific (i.e., they hybridize to DNA of a particular chromosome at lower c 0 t 1 2 than with DNA of other chromosomes; c 0 t , 2 being the time required for one half of an initial concentration (c 0 ) of probe to hybridize to its complement).
  • probes are feature (e.g., telomere, centromere, etc.) specific.
  • Such probes being proximal to the telomere, are capable of defining and identifying translocations that may be so close to the chromosomal termini as to be otherwise cryptic. Both types of probes may be used if desired. Sources of such probes are available from the American Type Culture Collection, and similar depositories.
  • the oligonucleotide probes used in accordance with the methods of the present invention are of a size sufficient to permit probe penetration and to optimize reannealing hybridization.
  • labeled DNA fragments smaller than 500 nucleotides in length, and more preferably of approximately 150-250 nucleotides in length, probes are employed.
  • Probes of such length can be made by synthetic or semi- synthetic means, or can be obtained from longer polynucleotides using restriction endonucleases or other techniques suitable for fragmenting DNA molecules. Alternatively, longer probes (such as polynucleotides) may be employed.
  • the oligonucleotide probes are synthesized so as to contain biotinylated or otherwise modified nucleotide residues. Methods for accomplishing such biotinylation or modification are described in U.S. Patent Nos. 4,687,732; 4,711,955; 5,328,824; and 5,449,767, each herein inco ⁇ orated by reference. Biotinylated nucleotides and probes are obtainable from Enzo Biochem, Boehringer Mannheim, Amersham and other companies.
  • biotinylated or otherwise modified nucleotides are produced by reacting a nucleoside or nucleotide with a mercuric salt under conditions sufficient to form a mercurated nucleoside or nucleotide derivative.
  • the mercurated product is then reacted in the presence of a palladium catalyst with a moiety (e.g., a biotin group) having a reactive terminal group and comprising three or more carbon atoms. This reaction adds the moiety to the purine cr pyrimidine ring of the nucleoside or nucleotide.
  • such modified probes are used in conjunction with competitor DNA in the manner described by Ward et al. (WO90/05789), herein inco ⁇ orated by reference.
  • Competitor DNA is DNA that acts to suppress hybridization signals from ubiquitous repeated sequences present in human and other mammalian DNAs.
  • alu or kpn fragments can be employed, as described by Ward et al. (WO90/05789).
  • probe DNA bearing a detectable label and competitor DNA are combined under conditions sufficient to permit hybridization to occur between molecules having complementary sequences.
  • two sequences are said to be able to hybridize to one another if they are complementary and are thus capable of forming a stable anti-parallel double- stranded nucleic acid structure.
  • Conditions of nucleic acid hybridization suitable for forming such double stranded structures are described by Maniatis, T., et al. (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)), by Haymes, B.D., et al. (In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985), and by Ried, T. et al. (Proc. Natl. Acad. Sci.
  • the sequences need not exhibit precise complementarity, but need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure. Thus, departures from complete complementarity are permissible, so long as such departures are not sufficient to completely preclude hybridization and formation of a double-stranded structure.
  • the quantity of probe DNA combined with competitor DNA is adjusted to reflect the relative DNA content of the chromosome target. For example, as disclosed by Ward et al. (WO90/05789), chromosome 1 contains approximately 5.3 times as much DNA as is present in chromosome 21.
  • a proportionally higher probe concentration would be employed when using chromosome 1 specific probes.
  • the resulting hybridization mixture is then treated (e.g., by heating) to denature the DNA present and is incubated at approximately 37 °C for a time sufficient to promote partial reannealing.
  • the sample containing chromosomal DNA to be identified is also heated to render it susceptible to being hybridized to the probe.
  • the hybridization mixture and the sample are then combined, under conditions sufficient to permit hybridization to occur. Thereafter, the detection and analysis of the hybridized product is conducted by detecting the fluorophore label of the probe in any of the methods described below.
  • probes are labeled either directly (e.g., with fluorescein) or indirectly (e.g., with biotinylated nucleotides or other types of labels), and permitted to hybridize to chromosomal DNA.
  • the hybridized complexes are incubated in the presence of streptavidin, that had been conjugated to one or more fluors. The streptavidin binds to the biotinylated probe of the hybridized complex thereby permitting detection of the complex, as described below.
  • One aspect of the present invention concerns the identification of a set of seven fluors that are be well resolvable by the excitation-emission contrast (EEC) method.
  • EEC excitation-emission contrast
  • multi-fluor combinatorial labeling depends in general on acquiring and analyzing the spectral signature of each object i.e., obtaining the relative weighting coefficients of the component fluors.
  • the method chosen was conventional bandwidth-restricted widefield imaging using epi- fluorescence triplets, viz. excitation filter, dichroic reflector and emission bandpass filter.
  • the limited spectral bandwidth available for imaging (roughly 380-750 nm), and the extensive overlap between the spectra of organic fluors, makes separating multiple fluors spectroscopically during the imaging step a significant technical challenge.
  • Contrast ratio plots were first computed for each of the fluors vs. its two neighbors. These plots indicate regions where pairwise contrast is high enough to be useful. A constraint on the practically attainable contrast is that regions of high contrast generally lie far down the flanks of at least one of the spectra i.e., where excitation and/or emission are strongly sub-optimum. Further, to attain the required degree of selectivity it is necessary to use filters of bandwidths in the range 5-15 nm (cf. approx. 50 nm for 'standard' filter sets). Together, these impose a severe sensitivity penalty. The goal of 10% maximum crosstalk represents an acceptable, practical compromise between sensitivity and selectivity.
  • the shift is a vector characteristic of each filter and its orientation in the epicube.
  • automatic compensation for image displacement is a necessary part of the processing software.
  • Manufacturing variations of a few nm in peak wavelength and FWHM specifications can have significant effects on the EEC ratios. Filter errors to long wavelength may be fine-tuned by tilting, but this option is severely curtailed in the case of emission filters because of increased image aberrations and worsened pixel shifts. There is no equivalent way to compensate short-wavelength errors.
  • the first member of the set of fluors is the counterstain DAPI, which gives a weak G-like banding pattern. Five of the remaining six fluors may be used combinatorially to paint the entire human chromosome set. All are available as avidin conjugates (for secondary detection of biotinylated probe libraries) or directly linked to dUTP (for direct labeling).
  • fluors comprise the preferred fluors of the present invention and are: 4'-6-diamidino 2-phenyl indole (DAPI), fluorescein (FITC), and the new generation cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
  • DAPI 4'-6-diamidino 2-phenyl indole
  • FITC fluorescein
  • new generation cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.
  • the abso ⁇ tion and emission maxima for the respective fluors are: DAPI (Abso ⁇ tion maximum: 350 nm; Emission maximum: 456 nm), FTTC (Abso ⁇ tion maximum: 490 nm; Emission maximum: 520 nm), Cy3 (Abso ⁇ tion maximum: 554 nm; Emission maximum: 568 nm), Cy3.5 (Abso ⁇ tion maximum: 581 nm; Emission maximum: 588 nm), Cy5 (Abso ⁇ tion maximum: 652 nm; Emission maximum: 672 nm), Cy7 (Abso ⁇ tion maximum: 755 nm; Emission maximum: 778 nm).
  • fluorescence is arguably the most powerful, because of its high absolute sensitivity and multiparameter discrimination capability.
  • Modem electronic cameras used in combination with high numerical aperture microscope objectives and state of the an optical filters are capable of imaging structures labeled with as little as a 10-100 fluor molecules per pixel.
  • fluor-tagged single-copy DNA sequences as small as a few hundred bases in size are detectable under favorable conditions.
  • the availability of families of spectrally distinguishable fluors makes simultaneous imaging of several different targets in the same specimen possible, either directly or through combinatorial or analog multiplex methods.
  • multi-fluor discrimination may be based on differential excitation of the fluors, differential emission, fluorescence lifetime differences, or on more complex but still analyzabie observables such as fluorescence anis ⁇ tropy.
  • This discussion assumes an epi-imaging geometry. Table 1 describes the symbols and operators relevant to the theoretical considerations of fluorescence.
  • J j ⁇ 5( ⁇ ,p). d ⁇ can be measured with a bolometric detector such as a calibrated micro-thermopile placed at or near the focal plane of the objective lens.
  • the S/N of each pixel increases indefinitely as (photons detected) 1 2 . How rapidly S/N increases depends on excitation strength, but the relationship between S/N and dose does not.
  • the effect of non-zero bleaching constant is to change the t 1 2 function to an asymptotic function, the form of which depends on the bleaching mechanism.
  • the asymptote once again does not depend at all on excitation rate, although the speed of approach to the asymptote does. If finite camera noise is added to photobleaching, it is found that the S/N climbs to a maximum value, then falls as the fluor is exhausted.
  • the microscope objective compresses the excitation beam, in a nonconfocal microscope it does not focus it to a point and so has no bearing on the fluorescence image resolving power (incoherent emitter).
  • the commonest excitation source for fluorescence microscopy is the high pressure short mercury arc, whose spectrum consists of pressure-broadened lines from the UV to the middle region of the visible spectrum (principal wavelengths are 334.1 nm, 365.6 nm, 404.7 mm, 435.8 nm, 546.1 nm. 577.9 nm), superimposed on a weaker thermal continuum. Many fluorophores have excitation spectra that overlap one or other of the mercury lines to an acceptable extent. Others (of which the best known is FTTC) do not, but may be adequately excited by the continuum if a wide enough excitation bandwidth is employed. Another common source is the high pressure xenon short arc, which produces an almost uniform continuum from ca. 300 nm: to beyond
  • nm 900 nm.
  • the power nm"' is almost everywhere less than the mercury continuum for the same arc wattage.
  • a high CW power or pulsed quartz halogen lamp outperforms the xenon beyond about 450 nm.
  • Certain fluors are well matched to laser excitation (e.g., Ar+ @488 nm for FITC, He-Ne @ 632.8 nm for Cy5, semiconductor diode - pumped YAG @ 680 nm for Cy5.5).
  • the excitation filter Fl and dichroic beamsplitter DB1 can usually be chosen to give adequate overlap between the source spectrum and the fluor excitation spectrum. If an arc line is available, the Fl bandpass need be no wider than the line. If not, and part of the thermal continuum must be used, the wider the Fl bandpass the greater will be the available excitation flux.
  • the goal of high excitation efficiency is generally secondary to the need to exclude excitation light from the emission path. This limits how close the excitation and emission bandpasses can be placed to one another, and hence constrains the excitation bandwidth. For fluors with small Stokes' shifts, high quality filters with very steep skirts are required.
  • the excitation filter must be rigorously 'blocked' on the long wavelength side, and have no pinholes, scratches, or light leaks around the edge.
  • Dichroic beamsplitters are currently much less 'evolved' than bandpass interference filters, meaning that the slopes of their transmit ⁇ -> reflect transitions are far less than the skirt slopes of premium notch filters, and there may be large spectral intervals where they oscillate between intermediate states of partial reflectance and transmittance.
  • the main pu ⁇ ose of a dichroic beamsplitter is to improve the combined efficiency of excitation and emission, rather than to define the wavelength response of the instrument.
  • the resolvability of overlapping fluors in imaging microscopy may depend critically on the degree of excitation contrast that can be achieved (see C, below).
  • the variation with wavelength of the ratio of the extinction coefficient of two fluors is the excitation contrast spectrum. It can readily be calculated from the digitized abso ⁇ tion spectra. Depending on the overlap of the abso ⁇ tions, their ratio spectrum may either show a distinct peak or may grow indefinitely large. In either case, it is usually possible to choose an excitation wavelength that favors one fluor over another to a useful extent (from a factor of 3-4 fold up to a hundredfold or more).
  • the detector pixel p accumulates signal (detected photons) at a rate:
  • G represents the efficiency with which the optics gather the fluorescence and transmit it to the detector; it may be assumed to be wavelength independent to first order.
  • NA numerical aperture
  • NA additionally determines the spatial resolving power of the microscope, because it scales the dimensions of the Eisenhofer diffraction pattern produced in the image plane by a point source in the specimen plane.
  • Several 'rules' are in use for specifying the resolving power of a lens, depending on how much overlap of the Airy discs of two adjacent objects is deemed to constitute the threshold of resolution.
  • image 'noise' at p is determined by the statistical variance in the number S(p) of fluorescent photons detected in time interval ⁇ t.
  • S(p) is an integral of the form:
  • bleaching of fluors in solution may be mechanistically and kinetically much more complex.
  • a common mechanism involves ring opening following peroxidation of the fluor excited state, e.g., by O2 or O2 2" .
  • This type of bleaching may be considerably slowed by rigorous deoxygenation or by the use of oxygen radical scavengers (i.e., antifade agents) such as tertiary amines (p- phenylene diamine or DABCO).
  • oxygen radical scavengers i.e., antifade agents
  • tertiary amines p- phenylene diamine or DABCO
  • a nonideal detector contributes noise of many kinds, detailed analysis of which may be intractable.
  • the simplest noise component is fluctuation in the so-called 'dark current,' i.e., the flow of thermally excited carriers within the detector. If this noise is assumed to be random, it adds to the photon shot noise in RMS fashion.
  • the mean photogenerated signal is F s ⁇ ' and the mean dark count is D s"'
  • the S/N after time ⁇ t is F.( ⁇ t) 1 2 / (F + D) 1 2 ; S/N still increases as ( ⁇ t) 1 2 , but more slowly than for a noiseless detector.
  • photobleaching is present, however, the situation is entirely different.
  • the principal design goals for a single-fluor imaging system are: 1. To achieve an adequate rate of excitation of the fluor (F). 2. To collect the fluorescence of F and image it onto the detector with high efficiency and with the necessary spatial resolution. 3. To prevent reflected and/or scattered excitation light from reaching the detector.
  • the dichroic beamsplitter transition wavelength is specified at for example, 20 mn to the red of the excitation passband. This ensures a high level of rejection of exciting light reflected and/or scattered from the specimen and/or microscope optics.
  • the emission filter cut-on is usually considerably steeper than the dichroic edge, and so can be placed practically coincident with it.
  • the most efficient emission filter is a long-pass element.
  • the preferred filter of this type is Schott glass, which transmits upwards of
  • the multiparametric imaging of the present invention not only increases the throughput of information about the system under observation and makes more efficient use of the biological material, but also can reveal spatial and temporal correlations that might otherwise be difficult to establish reliably.
  • two or more labels can be used combinatorially, which permits discrimination between many more object types than there are spectrally distinguishable labels.
  • Some examples of multi-fluor imaging are: a. The co-distribution of proteins in structures such as microtubule networks may readily be visualized using immunolabels linked to different fluors. b. Multiple genes may be simultaneously mapped by fluorescence in situ hybridization (FISH) to a single metaphase chromosome spread.
  • FISH fluorescence in situ hybridization
  • Such signals cannot usually be discriminated reliably on the basis of intensity alone, and are usually mo ⁇ hologically identical (diffraction-limited points). However, they are readily discriminated by discrete or combinatorial multi-fluor labeling.
  • Identification of small chromosomal translocations is most readily done by painting with chromosome specific DNA probe libraries linked to separable fluors, used either singly or combinatorially.
  • Analysis of mixed populations of mo ⁇ hologically identical bacteria can also be achieved using species-specific DNA or ribosomal RNA probes coupled to separable fluors.
  • the primary design goal of a multi-fluor imager is to spectrally resolve the fluorescence at any pixel location into components corresponding to each fluor.
  • Methods for spectrally resolving complex signals in fluorescent microscope images are outlined below. There are several ways to resolve spectrally complex signals, i.e., to determine which fluors contribute to the fluorescence at a given pixel location. The most general method in principle is to spectrally disperse the 2D image along a third axis, orthogonal to the x,y axes.
  • a solution is to extract the light corresponding to each object with a probe (e.g., a fiber optic) and disperse it with an imaging spectrograph onto a 1 -dimensional array detector.
  • a probe e.g., a fiber optic
  • an imaging spectrograph onto a 1 -dimensional array detector.
  • the most reasonable method for full spectral analysis in microscopy is to image through a variable narrow band filter. An image is recorded at each wavelength; intensity values at a given pixel location through the series represent a weighted emission spectrum that can be fit to a linear combination of the known spectra of the component fluors.
  • the coefficients are products of the relative molar amounts of the fluors with their extinction coefficients at the exciting wavelength and their fluorescence quantum yields. If the last two are known, the first is obtainable from the fit. In general, it is necessary to take several such image sets, at several excitation wavelengths, to get a unique fit. With enough iterations, this process generates a 3D surface of intensity values as a function of both excitation and emission wavelength.
  • This comprises a complete spectral signature of the pixel, giving a very highly constrained solution for the relative amounts of its component fluors.
  • the mole ratios could be mapped back onto the x,y coordinates of the image, with appropriate pseudocolor coding, to give a 'composition map'.
  • This general (and quantitative) method has a number of technical difficulties, although none of them is insurmountable. The first is that a large number of images are required to evaluate a single microscope field. Imaging time is long, and extensive differential photobleaching of the fluors would make it impossible to achieve a self consistent "fit" to the spectral data. Instrument stability is also an issue, particularly with arc sources, the output spectra of which change throughout their life. Finally, the amount of computation required to generate a composition map would realistically limit the analysis to small image regions only.
  • excitation-emission contrast (EEC) approach is in principle applicable to analysis of images involving multiple fluors with fine-grained distributions of mole fraction (e.g., fluorescence ratio imaging), subject to image S/N and the limitations of differential bleaching rates and source instability.
  • EEC excitation-emission contrast
  • this is not achievable on the basis of excitation contrast or emission contrast alone. Simultaneous selection on the basis of both excitation and emission are required.
  • excitation and emission optics that have no wavelength selectivity cannot be used, because the excitation light scattered into the detector would overwhelm the fluorescence by several orders of magnitude.
  • One solution is to use multiple-bandpass filters designed for the specific set of fluors to be used.
  • the excitation filter defines narrow passbands that overlap the fluor excitation spectra.
  • the emission filter defines similar passbands that interdigitate between the excitation bands and overlap the fluor emission spectra (the reddest fluor could use a long-pass filter).
  • the dichroic beamsplitter alternates between reflect (overlapping the excitation passbands) and transmit (overlapping the emission passbands).
  • a second theoretical solution would be to excite at a wavelength where all the fluors absorb. This is often possible because many fluors are excitable to states higher than S 1 using photons in the middle UV, but because of internal relaxation processes give 'normal' fluorescence. For example, many laser dyes can be excited at the nitrogen laser wavelength, 337 nm, far to the blue of their visible absorbances. It would be straightforward to block such exciting light from the emission path, using a long-pass filter (e.g., 380 nm), while allowing all fluorescences to simultaneously reach the detector.
  • Drawbacks to the use of UV excitation include increased rates of photochemical decomposition of the fluor, and the expense of suitable UV optics. Thus, the method has not found widespread use.
  • the multi-bandpass method has the limitation that construction of multiple bandpass elements giving adequate contrast between more than 3 fluors is extremely difficult.
  • a generally more powerful approach is to construct optimized filter sets for each fluor, and switch them as needed.
  • the primary goal on the excitation side is high excitation flux, to give a bright image.
  • fluors may be imaged by sequentially switching filters that are designed using the same criteria as single-fluor sets (except that long-pass emission filters are proscribed for all but the longest- wavelength fluor). Crosstalk of a few percent is usually allowable, and can be compensated numerically if necessary.
  • the principal technical problem with serial imaging is image displacement when filters are changed i.e., the coordinate systems of the individual members of an image series are not in precise registration. This problem arises from nonidealities in the emission channel optics.
  • Two displacement components can be identified: i. a reproducible offset that is unique to each filter set. This component is a fixed vector, and arises mainly from imperfect parallelism (i.e., wedging) between the top and bottom faces of the emission bandpass filter. There is also a small component due to wedging in the dichroic beamsplitter, but since this element is very thin the effect is minor. Since the wedging vector is a constant for each filter set, it can be automatically removed in the computer.
  • the size of the offset can also be reduced to very small values ( ⁇ 0.1 ⁇ ) by selecting emission filters for a high degree of parallelism e.g., by measurement in a laser autocollimator. ii. a random component due to vibration and hysteresis in the filter switching mechanism. The magnitude of the noise depends on the filter switching mechanism. The worst are manual push-pull sliders, particularly when the operator actuates them using uncompensated forces. The best are motorized filter cassettes, in which all mechanical torques act against the microscope body.
  • the detection of fluor is accomplished using optical filters, in a modification of the method of Ried, T. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 89: 1388-1392 (1992), herein inco ⁇ orated by reference).
  • DAPI 4' ,6-diamidino-2-phenylindole
  • DAPI has very broad excitation and emission spectra, and a very large Stoke's shift.
  • the fluorescence of DAPI peaks to the red of Cascade
  • DAPI emits the bulk of its fluorescence to the red of CB.
  • the wavelength of maximum emission contrast for DAPI vs. both CB and FTTC is 490 nm.
  • a suitable imaging-grade filter is the Omega 485DF22. There are no mercury lines within its bandpass, so good
  • DAPI images with low flare are expected.
  • the calculated emission contrast between DAPI and both Cascade Blue and FITC is 6.8.
  • the overall contrast achievable for DAPI vs. Cascade Blue is approximately 27-fold.
  • the overall contrast of DAPI vs. FTTC is very much higher than this, because at the DAPI excitation wavelength the excitation of FTTC is close to zero.
  • the Omega 450DRLP02 dichroic beamsplitter is very well matched to the proposed excitation and emission filters.
  • Cascade Blue has a broad, two-peak excitation spectrum that overlaps
  • the absorbance peak of Cy3 is at 551 nm, at which wavelength the excitation of FTTC is essentially zero (contrast parameter Rb jumps to extremely high values to the red of 525 nm).
  • the Cy3 extinction peak overlaps strongly with the Hg 546.1 nm line.
  • the excitation contrast ratio for Cy3/Cy3.5 is everywhere small, and varies weakly with wavelength. At 551 nm, the absolute value of the excitation contrast for Cy3 vs. Cy3.5 is less than 2, and it only rises significantly far to the blue where the Cy3 absorbance is very low and FTTC absorbance is high.
  • Cy3.5 relative to Cy5 is also quite large (absolute value approximately 8.0).
  • the emission contrast parameter for Cy3.5 vs. Cy3 is small at all wavelengths where the Cy3.5 emission is usefully strong, i.e., isolation of Cy3.5 from Cy3 must rely mainly on excitation contrast.
  • the emission contrast for Cy3.5 vs. Cy5 is also large over a considerable spectral interval (and rises to very high values below 640 nm). This permits a fairly broadband filter to be used to image Cy3.5; a suitable element is the Omega 615DF45. Almost no bleadthrough of either Cy3 or Cy5 into the Cy3.5 channel is expected with the above combination of excitation and emission conditions.
  • the Omega 590DRLP02 is a suitable dichroic for this channel.
  • the "official" filter for exciting Cy5 in this way is the Omega 640DF20, which will give an excitation contrast with Cy5 of about 1.8. d.
  • a much brighter source for exciting Cy5 is the He-Ne laser (632.8 nm). It does not, however, improve excitation contrast vs. Cy5.5.
  • the emission contrast for Cy5 vs. Cy3.5 peaks at 673 nm, just to the red of the fluorescence intensity peak.
  • the closest available filter is the Omega 660DF32 where the emission contrast ratio is approximately 3.1. This compounds the very high excitation contrast for Cy5 vs. Cy3.
  • the emission contrast for Cy5 vs. Cy5.5 goes to very large values at wavelengths shorter than 675 nm.
  • the Omega 660DF32 filter is ideally set up to take full advantage of this. Unfortunately, it is uncomfortably close to the 640DF20 exciter, so some flare from reflected/scattered excitation light is to be expected. Use of the He-Ne laser would remove this problem, g.
  • the best available dichroic beamsplitter for Cy5 imaging is the Omega 645DRLP02, particularly if a He-Ne is used as the excitation source.
  • CY5.5 is the penultimate dye of the set, and is very well separated from Cy7. Thus, only its contrast relative to Cy5 need be considered in detail: a.
  • the contrast parameter Rt ⁇ for the Cy5.5/Cy5 pair rises to large values to the red of 670 nm. Thus, it is possible to achieve very high excitation contrast for this pair of fluors (analogously to Cy3.5/Cy3).
  • the Hg arc is a poor source for exciting Cy5.5.
  • the best available source is a 680 nm diode-pumped frequency doubled YAG microlaser (Amoco), which coincides with the peak of the Cy5.5 absorbance. At 680 nm, the excitation contrast ratio for Cy5.5/Cy5 is 5. 1.
  • a suitable excitation filter is the Ealing 35-4068. c.
  • the emission contrast ratio between Cy5.5 and Cy5 peaks at 705 nm, approximately 3 nm to the red of the Cy5.5 intensity curve.
  • the numerical value for Sfo at this point is 4. If the Omega 700EFLP longpass emission filter is used, a contrast ratio of approximately 3 (averaged out to 800 nm) is expected. This, combined with the high excitation contrast, makes imaging
  • Cy7 is the reddest dye of the set.
  • the excitation and emission spectra are well separated from Cy5.5, and are well matched to the Omega 740DF25/770DRLPO2/780EFLP triplet.
  • the Oriel 58895 is an appropriate IR blocker for Cy7.
  • Filters selected for imaging the DAPI, FITC, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 fluor set are summarized in the Table 2 below. None of these filter sets correspond to the filter sets supplied by manufacturers of conventional fluorescence microscopes as narrow band excitation and fluorescence detection is mandatory to achieve sufficient contrast.
  • the filter sets were selected on the basis of maximum spectral discrimination rather than maximum photon throughput. Image exposure times were varied to adjust for photon flux differences and flux excitation cross-sections. Narrow band excitation and fluorescence detection is mandatory to achieve sufficient contrast. Appropriate excitation and emission filter sets were used to optically discriminated these fluors (Table 2).
  • the combinatorial labeling strategy relies on accurate measurements of intensity values for each fluorophore. Critical features are accurate alignment of the different images, correction of chromatic aberrations, and specific quantitation of each fluorophore. Because simple manual image manipulation could not realize these demands new software was developed in our lab.
  • This program comprises the following steps in sequential order: (1) correction of the geometric image shift; (2) calculation of a DAPI segmentation mask; (3) for each combinatorial fluor, calculation and subtraction of background intensity values, calculation of a threshold value and creation of a segmentation mask; (4) use of this segmentation mask of each fluor to establish a "Boolean" signature of each probe; (5) for each chromosome, display of the chromosome material next to the DAPI image; (6) create a composite gray value image, where each labeled object is encoded with a unique gray value; (7) final presentation of the results using a look-up-table (LUT) that assigns each gray value a particular color
  • the above-described program was developed on the basis of an image analysis package (BDS-Image) implemented on a Macintosh Quadra 900.
  • Image shifts caused by optical and mechanical imperfections were corrected by the alignment of the gravity center (center of mass) of a single chromosome in each image according to a procedure described by Waggoner, A. et al. (Methods Cell Biol 50:449-478 (1989)) and modified (du Manoir, S. et al., Cytometry 9:4-9 (1995); du Manoir, S. et al., Cytometry 9:21-49 (1995), all herein inco ⁇ orated by reference).
  • the DAPI image was used to define the mo ⁇ hological boundary of each chromosome.
  • selective excitation of multiple fluors and analysis of fluorescence spectral signatures can be carried out using dispersion optics rather than wavelength-selective transmission filters.
  • Such optics may be used to create filters of any passband characteristic, including short-pass, long-pass, single bandpass and multiple bandpass functions.
  • a dispersion element (prism or grating) is used in conjunction with a wavelength-selective spatial filter to create the desired spectral response.
  • the combination is referred to herein as a "comb filter.”
  • a comb filter the spectral distribution of the exciting light may be tailored for optimum simultaneous excitation of multiple fluors.
  • the inverse comb filter may also be used to selectively block from the CCD camera only the wavelengths used for excitation: the remaining wavelength intervals (corresponding to the gaps between teeth of the comb) are available for spectral analysis of the fluorescence emitted by the fluors. This analysis constitutes the spectral signature.
  • an interferometer may be used in conjunction with an epi-fluorescent microscope.
  • a light source for excitation of fluorescence that is either coherent (e.g. an Argon laser) or incoherent (e.g. a
  • Mercury arc lamp may be used.
  • a Mercury-Xenon mixed gas arc lamp is preferred due to its intense Mercury lines and broad Xenon visible and near-infrared continuum.
  • the Sagnac interferometer has a larger acceptance angle, greater claritye, and is less sensitive to alignment, vibration, and temperature variations than a similar Michelson interferometer.
  • the Sagnac interferometer is a common path interferometer.
  • An interferometer consists of two or more interfering beams of light. In a common path interferometer there are two beams each traveling the same path but in opposite directions.
  • the optical paths are produced by reflecting light through beamsplitters, for example.
  • Multiple beam interferometers operate by dividing the optical energy from a light source into two substantially equal beams of light. The two beams of light are combined after one is permitted to pass through a sample and the interference pattern (the changes in intensity of the combined light caused by the interference of two beams) is detected.
  • the light source is also divided into two substantially equal parts. Changing the angle of incidence of light on the beamsplitter, (by rotation of the interferometer, or rotation of an optic, such as a galvanometer driven mirror within the interferometer) causes the optical path length to be changed along one optical axis of the interferometer. This produces a fringe pattern along one axis of the detector, for example a CCD detector. The other axis of the detector can sample gray scale.
  • an advantage of the Sagnac interferometer is that it produces an optical path difference across an entire field of view, rather than at a single point.
  • w/a tan 30°
  • w is the aperture width of the interferometer
  • a is the length of each leg
  • n is the index of refraction of the interferometer glass.
  • the interference pattern or interferogram is most preferably detected with a CCD camera (such as a Princeton Instruments frame transfer CCD camera) capable of 512 X 512 pixels or larger. Since the interferogram in a Sagnac interferometer has an angular dependence, each pixel of the CCD detector measures a small interval of the interferogram. The fringe spacing of the interferogram is set such that a pixel on the CCD detector can adequately sample the interferogram.
  • OPD Optical Path Difference
  • ⁇ m i n is the shortest wavelength in the spectrum to be measured by the interferometer.
  • This OPDpi xe j determines the theoretical limit of the resolving power of the interferometer.
  • the interferogram is being moved across the CCD detector, such that the maximum optical path difference is then given by the relation
  • OPD rnax N(OPD pixel )
  • N is the linear dimension of the CCD detector in pixels.
  • Each angular displacement of the light incident on the interferometer beamsplitter may then correspond to one or several OPDpj xe i' s . And, in the case of a CCD detector, one frame of CCD data is required to sample this angular displacement.
  • each pixel comprises an interferogram which contains within it information about the spectrum of light falling on the pixel, the intensity of light falling on that pixel, and the x and y coordinates of the pixel.
  • the spectrum of light may be recovered from the interferogram by the use of a computational Fourier
  • the light used to excite the fluorescence must be blocked from entering the interferometer.
  • This excitation light is often 10 8 to 10 12 more intense than the fluorescence that is emitted from the sample. Without blocking by using optical filtering, this excitation light would saturate the CCD. However, this filter need only be fabricated so as to block the excitation, all other wavelengths may be allowed to pass.
  • ultra violet (UV) light is used to excite the fluorescent probes.
  • the UV light may be easily blocked with a long pass interference filter allowing the visible and near-infrared colors to pass through to the interferometer.
  • This embodiment has the advantage that UV will excite many of the fluorescent dyes currently in use.
  • This embodiment also has the advantage that it will allow better than 90% transmittance of the visible fluorescence to the interferometer.
  • the disadvantage of UV is that it photobleaches the dyes faster than visible light.
  • Both the input and the output lens of the interferometer are preferably very high efficiency camera lenses, and do not significantly effect the efficiency of imaging.
  • the focus of the image within the interferometer is most preferably adjusted so as to be constant for the variable powers of the zoom eyepiece, and thus a microscope having the characteristic of infinite image distance (such as Olympus AX70 microscope) are preferred.
  • the above-described interferometer possesses certain advantages over optical filters.
  • One key advantage is that all the light emitted by fluorescence is theoretically available for detection, whereas the transmittance of an interference filter is limited.
  • Another advantage is that since the filters do not have to be changed, there is no image shift due to the non-parallelism of filters.
  • FISH Fluorescent In situ Hybridization
  • cytogenetic diagnosis of genetic disease such as the pre- or post-natal diagnosis of disease, complex tumor karyotyping, the analysis of cryptic translocations (especailly through the use of telomere-specific probes). It provides a novel method for automated chromosome identification and analysis.
  • diseases prenatal disease, cancers (especially BRCA1 or BRCA2 associated breast cancer), leukemias, Down's Syndrome, etc.) are characterized by rearrangements and other chromosomal abnormalities that can be discerned using the methods of the invention.
  • Chromosome karyotyping by conventional cytogenetic banding methods is both time consuming, expensive and not easily automated.
  • the detection of recurring genetic changes in solid tumor tissues by karyotyping are particularly problematic because of the difficulty in routinely preparing metaphase spreads of sufficient quality and quantity and the complex nature of many of the chromosomal changes, which make marker chromosome identification based solely on banding patterns extremely difficult.
  • attempts to automate karyotype analysis over the past twenty years e.g., pattern matching, eigen analysis
  • FISH karyotype Advantages of the FISH karyotype are the instant identification of the chromosomal origin of marker chromosomes, double-minutes and homology staining regions ("HSRs"). Even “poor quality" chromosome spreads can be evaluated.
  • HSRs homology staining regions
  • the development of specific probe sets that stain particular regions of chromosomes (e.g. telomeric regions) for the identification of cryptic translocations would overcome limitations of the whole chromosome painting probes.
  • such probes could be used to generate multicolor "barcodes" on individual chromosomes thereby facilitating the automated analysis of karyotype.
  • Probes can also be designed that would be specific for a particular arm of a chromosome, thereby permitting a molecular characterization of translocation breakpoints, hot spots of recombination, etc.
  • Other applications would include rapid evolutionary studies, provided that the protocols for multicolor FISH on human chromosomes can be adjusted, as expected, for applications on other species.
  • the methods of the invention may also be used to assess the presence or absence of infectious agents (treponema pallidum, rickettsia, borrelia, hepatitis virus, HTV, influenza virus, he ⁇ es, Group B streptococcus, diarrhea-causing agents, pathogens causing acute meningitis, etc.) in tissue, or in blood or blood products.
  • infectious agents such as leptin, leptin, leukin, etc.
  • This can be accomplished by employing labeled probes specific for such agents.
  • serotype-specific probes permit the rapid serotyping of such agents, or the determination of whether any such agents carry drug resistance determinants.
  • the methods of the present invention may be used to assess chromosomal abnormalities caused by exposure to radiation (such as personnel exposed to the radioactivity of nuclear power plants).
  • the methods of the present invention may be used to quantitate microorganisms that are difficult to propagate (such as anaerobic microorganisms involved in periodontal disease).
  • the methods of the present invention provide a means for the rapid diagnosis of acute bacterial meningitis.
  • serotype-specific probes to perform serological analysis
  • probes that are specific to particular drug resistance determinants and thereby rapidly determine not only the presence and identity of an infectious agent, but also its susceptibility or resistance to particular antibiotics.
  • the methods of the present invention further permit simultaneous mapping of a large number of different DNA probes.
  • the analysis of chromosomal number and architecture in individual intact cells becomes accessible.
  • Inte ⁇ hase cytogenetics is already possible with small region specific probes, e.g. YAC-clones.
  • the accuracy of such analysis could be increased by a three dimensional analysis using a laser scanning microscope, or more preferably, a CCD camera system with a Z-axis stepping motor coupled with 3-dimensional (3-D) image deconvolution software.
  • Suitable 3-D image deconvolution software is obtainable from Imstar Co ⁇ . (Paris, France) or Scanalytics, Inc. (Boston).
  • the methods of the present invention enable one to examine chromosome architecture or quantitate the chromosome contents of nuclei in single hybridization experiments. Questions relating to intranuclear chromosomal organization as a function of developmental status, cell cycle or disease state can accordingly be addressed.
  • the ability to quantitatively assess the levels of multiple mRNAs or proteins in a single cell or to determine if they exhibit different intracellular distributions could prove extremely useful in addressing a myriad of interesting biological questions.
  • the multiparametric imaging of the present invention does not merely increases the throughput of information, it also makes more efficient use of the biological material. Thus, it can reveal spatial and temporal correlations as well as mosaicisms that might otherwise be difficult to establish reliably.
  • the chromosomes of a particular karyotype are pseudo-colored to thereby facilitate the assignment of the chromosomes, or the recognition of translocations, deletions, etc.
  • the digitized images of the chromosomes may be stored in a computer-readable storage device (such as a magnetic or optical disk) to facilitate their comparison with other chromosomal images or their transmission and study.
  • probes may be employed that are translocation specific or specific to sub-chromosomal elements or regions, such that the pseudocolorized chromosomes or chromosomal elements are displayed to the investigator as chromosomal images depicting a cytogenetic banding pattern (for example, the cytogenetic banding pattern of the metaphase chromosomes of the patient).
  • the position and sizes of individual bands is preferably digitized and stored so that an image of the chromosome may be stored on a computer.
  • the precise position of any translocation or other karyotypic abnormality can be discerned and stored.
  • the methods of the present invention thus permit karyotypic analyses to be conducted more widely and more accurately than was previously feasible.
  • the present invention may thus be used to systematically correlate karyotypic abnormalities with disease or conditions.
  • karyotypes of asymptomatic individuals can be obtained and evaluated in light of any subsequent illness (e.g., cancer, Alzheimer's disease, etc.) or condition (e.g., hypertension, atherosclerosis, etc.) in order to permit a correlation to be made between a patient's karyotype and his or her predisposition to different diseases and conditions.
  • karyotypes of individuals having diagnosed diseases or conditions can be obtained and evaluated in light of the extent of any subsequent progression or remission of the disease or condition so as to permit a correlation to be made between a particular karyotype and the future course of a disease or condition.
  • a computer or other digital signal analyzer may be employed to orient and arrange the chromosomal images as well as assigning and identifying the chromosomes of the karyotype.
  • a computer or other data processor will, upon assigning a particular chromosome to a particular designation (for example, upon assigning that a particular chromosomal image is the image of the chromosome 7 of the karyotype being evaluated), group the assigned chromosome with its homologue (e.g., the second chromosome 7 of the patient's karyotype) and generate, via a printer, monitor, or other output means, an ordered array of chromosomal images (such as one in which each autosomal chromosome is paired with its homologue, and in which the sex chromosomes X and Y are paired together).
  • the chromosomal images of such arrays will be the pseudocolorized images discussed above.
  • such psudocoloring may be internal to the process of assigning chromosomal identity, and not displayed in the output of the computer or digital signal analyzer. Rather, in this sub-embodiment, the output generated will be the light-microscope visible cytogenetic banding pattern of the metaphase chromosomes of the patient whose karyotype is being evaluated.
  • a scale in Morgans or other suitable units
  • Example 1 Combinatorial Labeling of Chromosomes
  • chromosome painting probes representing the 22 autosomes and the two sex chromosomes were used.
  • the DNA probes used were generated by microdissection.
  • Microdissected probes (National Center for Human Genome Research, Bethesda, MD) give a very uniform labeling of the target region.
  • the detailed protocols for microdissection and PCR amplification are described by Telenius et al. (Telenius, H. et al, Genes, Chromosomes & Cancer 4:251 -263 (1992); Telenius, H. et al, Genomics 75:718-725 (1992); Meltzer, P.S. et al, Nature Genetics 7:24-28 (1992); Guan, X.Y. et al, Hum Mol Genet 2: 1117-1121 (1993); Guan, X.Y.
  • the first member of the set of preferred fluors, DAPI was used as a general
  • the probes were subjected to a PCR amplification and labeled by nick translation.
  • Fluorescein (Wiegant, J. et al, Nuc Acids Res 79:3237- 3241 (1991)), Cy3, and Cy5 were directly linked to DUTP for direct labeling.
  • Cy3.5 and Cy7 were available as avidin or anti-digoxin conjugates for secondary detection of biotinylated or digoxinigated probes. They were synthesized using conventional N- succinamide ester coupling chemistry. For each probe one to three separate nick translation reactions were necessary, each with a single labeled fluor-labeled triphosphate or biotin or digoxigenin (Table 3).
  • probes labeled with equal amounts of different fluors did not give equivalent signal intensities for each fluor reflecting the fact that the filter sets were selected to maximize spectral discrimination rather than photon throughput.
  • probe concentrations for the hybridization mix had to be established carefully in a large number of control experiments. Hybridization conditions were optimized for these multiplex probes.
  • probes were denatured and hybridized for two to three nights at 37 °C to metaphase chromosome spreads in a conventional 50% formamide hybridization cocktail. The slides were washed at 45 °C in 50% formamide/2 x SSC three times followed by three washes at 60 °C in 0. 1 x SSC to remove excess probe.
  • Figure 1 provides a schematic illustration of the CCD camera and microscope employed in accordance with the present methods.
  • Figure 2 shows the raw data from a karyotypic analysis of chromosomes from a bone ma ⁇ ow patient (BM2486). Adjacent to each source image is a chromosome "mask" generated by the software program.
  • panels A and B are the DAPI image and mask
  • panels C and D are FTTC image and mask
  • panels E and F are Cy3 image and mask
  • panels G and H are Cy3.5 image and mask
  • panels I and J are Cy5 image and mask
  • panels K and L are Cy7 image and mask.
  • Figures 3A and 3B show the identification of individual chromosomes by spectral signature.
  • Figure 3A is the same photograph as Figure 2, except that it is gray scale pseudocolored.
  • Figure 3B displays the karyotypic array of the chromosomes.
  • the exceptional power of the methods of the present invention are illustrated by the ease with which the translocation of chromosomes 5 and 8 are identified in Figures 3A and 3B, relative to conventional non-chromosome specific karyotype analysis.
  • In situ hybridization has been recognized as having potential application as a means of analyzing individual bacterial cells in the absence of culture.
  • DeLong, E.F. et al. (Science 245: 1360-1363 (1989)), for example, used oligonucleotide probes complementary to the 16s ribosomal RNA (rRNA) sequences to differentiate an archaebacterium (Methanosarcina acetivorans) from the eubacteria Bacillus megaterium and Proteus vulgaris.
  • rRNA ribosomal RNA
  • the M-FISH methods of the present invention are used used to speciate bacteria involved in the microbial etiology of periodontal disease.
  • Several methods have previously been used to establish the microbial etiology of periodontal disease.
  • Well over 200 bacterial species can be identified in one sublingual plaque sample using labor-intensive cultural methods (Dzink, J.L. et al, J. Clin. Periodont. 72:648-659 (1985); Moore, W.E.C.; J. Periodont. Res. 22:335-341 (1987); Moore, W.E.C., Infect. Immunity 58:651-667 (1987)).
  • a test for in situ hybridization specificity is conducted using a mixture of bacteria that are mo ⁇ hologically distinct and non-cross hybridizing. Using such a mixture, one can immediately differentiate positive hybridization from that of non-specific binding by correlating the correct mo ⁇ hology with the correct probe.
  • Fusobacterium nucleatum (25586), Porphyromonas gingivalis (33277), Eikenella corrodens (23834), Prevotella intermedia (25611), and Actinobacillus actinomycetemcomitants (29522) were obtained from the American Type Culture Collection, Bethesda, MD, and Capnocytophaga species, C. ochracea (C25) and C. gingivalis (DR2001) were obtained from The National Institute of Dental Research, Bethesda, MD. Total genomic DNAs were isolated from F. nucleatum, P. gingivalis, E. corrodens, P. intermedia, A. actinomycetemcomitans, C. gingivalis, and C.
  • Total genomic bacterial DNAs are labeled by nick translation using biotin- 11 - dUTP (BIO), digoxigenin-11-dUTP (DIG) or dinitrophenol-11-dUTP (DNP) as described by Lichter, P. et al, Hum. Genet. 80:224-234 (1988), herein inco ⁇ orated by reference). Uninco ⁇ orated nucleotides are removed using a Sephadex G-50 spin column equilibrated with lOmM Tris-HCI 1 mM EDTA/0.1% SDS, pH 8.0. Labeled DNAs (2-6 ⁇ g) are ethanol precipitated and redissolved in 100% deionized formamide. F.
  • nucleatum DNA is labeled with BIO-11-dUTP (BIO) and detected with Cascade Blue avidin;
  • BIO BIO-11-dUTP
  • Cascade Blue avidin A. actinomycetemcomitans DNA is labeled with DNP-1 1-UTP (DNP) and detected indirectly with an FTTC conjugated antibody;
  • £. corrodens DNA is labeled with DIG-11-dUTP (DIG) and detected with a Rhodamine labeled anti-DIG antibody.
  • the bacteria are hybridized simultaneously with the three differentially labeled genomic DNAs.
  • Each sample is hybridized using 15 ⁇ l of hybridization solution which contains 50 ng of labeled probe and DNAse-treated salmon sperm DNA (15 ⁇ g) in 50% (vol/vol) deionized formamide/2xSSC (0.3 M sodium chloride/0.03 M sodium citrate, pH 1.0)15% dextran sulfate.
  • the solution is applied to the sample, covered with a coverslip and sealed with rubber cement. Both bacterial DNA and labeled DNA probe are denatured by heating at 80°C for 5-8 minutes in a oven. These steps are sufficient to permeabilize the bacteria for probe assessability.
  • the DNAs were allowed to reassociate by incubating the slides at 37°C for 18 hrs in a moist chamber. Posthybridization washings, blocking and detection were as described by Lichter et al. (7). Briefly, the slides were washed 3X in 50% formamide/2XSSC at 42°C for 5 minutes, and then washed 3X in 0.1XSSC at 60°C for 5 minutes. The slides were then incubated in a solution of 3% bovine serum albumin/4XSSC (blocking solution) for 30 minutes at 37°C.
  • Biotinylated probes were detected using fluorescein isothiocyanate-(FTTC)- avidin DCS (Vector Laboratories, Burlingame, CA, 5 ⁇ g/ml) Cascade Blue-avidin (Molecular Probes Inc., Eugene, OR, 10 ⁇ g/ml)) or Rhodamine-avidin (Boehringer Mannheim, Indianapolis, IN, 10 ⁇ g/ml).).
  • Digoxigenin-labeled probes were detected using FTTC or Rhodamine conjugated sheep anti-digoxigenin Fab fragments (Boehringer Mannheim, 2 ⁇ g/ml).
  • Dinitrophenol labeled probe was detected by incubating with rat anti-DNP antibodies (Novagen, Madison, Wl, 1:500 dilution)) and then with goat anti-rat FTTC-conjugated antibodies (Sigma, St. Louis, MO, 1 ⁇ g/ml).
  • bacterial DNA was counterstained with 4,6-diamidino-2- phenylindole (DAPI) at a concentration of 200 ng/ml.
  • DAPI 4,6-diamidino-2- phenylindole
  • the fluorochrome-conjugated antibodies or avidin are diluted into a solution of 4xSSC/l% BSA and 0.1% tween-20 (200 ⁇ l/slide) and incubated with the sample at 37°C for 30 minutes in the dark. The slides are then washed 3 times in 4xSSC/0.1% tween-20 at 42°C prior to viewing via epifluorescence microscopy.
  • Epifluorescence microscopy is conducted using a Zeiss Axioskop-20 wide- field microscope with a 63x NA 1.25 Plan Neofluar oil immersion objective and a 50W high pressure mercury arc lamp. Images are projected with a Zeiss SFL-10 photo-eyepiece onto a cooled charged-coupled device (CCD) camera (Photometries CH220; 512x512pixel array). Effective magnification is set by changing the microscope-camera distance, using a bellows. The 8-bit greyscale images are recorded sequentially using DAPI, FTTC and Rhodamine filter sets (manufactured by C. Zeiss, Inc., Germany) to minimize image offsets.
  • CCD charged-coupled device
  • FIG. 4 shows the differentiation of bacteria by in situ hybridization.
  • Panels A-E represent in situ hybridization assay results on a laboratory derived mixture of three bacteria (F. nucleatum, A. actinomycetemcomitans and E. corrodens) using a mixture of genomic DNA probes for each organism.
  • Panel A shows all the bacteria present in the microscope field detected by (DAPI), a general DNA binding fluorophore.
  • F. nucleatum is seen using Cascade Blue detection (panel B), A. actinomycetemcomitans with FTTC detection (panel C) and E. corrodens using Rhodamine detection (panel D).
  • Panel E is a computer-merged composite of panels B-D showing the differentiation of each bacteria in the sample. Note that a single A.
  • actinomyetemcomitans cell is detected in a clump of E. corrodens (panel ⁇ ) that could not be identified by DAPI staining (panel A).
  • Panel F is a similar analysis of a mixture of C. gingivalis (Rhodamine) and P. intermedia (FTTC) when hybridized with a combination of genomic DNA probes for those organisms.
  • Panel G shows an in situ hybridization assay for a combination of seven different bacteria hybridized with a mixture of seven genomic DNA probes.
  • F. nucleatum (1), E. corrodens (2) and A. actinomycetemcomitans (3) are shown in blue (Cascade Blue detection).
  • ochracea (5) are shown in red (Rhodamine detection), whereas P. intermedia (6) and C. gingivalis (7) are seen in yellow (FTTC detection).
  • Phase contrast microscopy was used to assist in determining mo ⁇ hological differences between ⁇ . corrodens and A. actinomycetemcomitans which were not readily evident through the hybridization generated signals.
  • Panel H represents an in situ hybridization assay for the presence of A. actinomycetemcomitans (FTTC detection) in a plaque sample obtained from a patient with localized juvenile periodontist.
  • FIG. 4 panel F shows a combined analysis of P. intermedia (BIO label and FTTC detection) and C. gingivalis (DIG label and Rhodamine detection).
  • ochracea group is seen with Rhodamine (DIG detection) and these bacteria are numbered 4 and 5 respectively; the C. gingivalis, P. intermedia group is identified with FTTC (DNP detection) and they are numbered 6 and 7 respectively. All seven bacteria are clearly differentiated using the combination of hybridization fluorescence and mo ⁇ hology. While mo ⁇ hological identification could be made simply on the basis of size, shape and color of the hybridization signal, more definitive mo ⁇ hological characterization is made by examination of the sample using phase-contrast optics.
  • the method provides a quantitative assessment of specific microorganisms without the necessity of culturing.
  • the technique can be applied to samples containing only a limited number of organisms and a single hybridization-positive bacteria can be detected quite easily in a 10 4" fold excess of hybridization-negative bacteria.
  • bacterial specified that are not clearly identified by a non-specific analysis (i.e., DAPI staining of DNA, Figure 4, panel A) can be detected by the hybridization-specific assay.
  • the intensity of the hybridization signal is extremely strong using either whole genomic DNA or subtracted (suppressed) probes.
  • data is collected by digital imaging, hybridization signals can be detected readily by eye and recorded by conventional photographic methods.
  • the sensitivity and photon counting capabilities of CCD-camera based imaging system offer considerable advantages for further refining microbial analysis by in situ hybridization. Detection of single copy sequences for specific toxin or drug resistance genes in individual bacteria should be feasible; individual human and murine genes have been visualized, both in metapha ⁇ e chromosomes and inte ⁇ hase nuclei by FISH (Lichter, P. et al, Hum. Genet. 80:224- 234 (1988)). The level of cross-homology between bacterial strains also can be rapidly assessed by quantitating the photon output of the hybridization signal from individual bacteria in a specimen. Finally, and most importantly, digital imaging is essential to fully exploit the potential of combinatorial fluorescence.
  • total genomic DNA can often provide adequately specific probes, thus eliminating the usual cloning and screening efforts necessary for probe production.
  • suppression hybridization techniques can be used to squelch cross- hybridization between organisms that share partial sequence homology.
  • C. orchracea and C. spumblea exhibit 22-23% sequence homology (Zambon, J. et al, J. Periodontal. 54:101 (1983)) yet specific hybridization to C. orchracea can be obtained with genomic DNA by preannealing the C. ochracea probe with an excess of C. spumblea DNA just prior to the in situ hybridization reaction.
  • genomic DNAs from seven anaerobic and facultative oral bacteria recognized as pathogens in periodontal disease are labeled non-isotopically and hybridized in situ to reconstructed bacterial mixtures.
  • Each probe is hybridized uniquely to the bacterium from which it was derived.
  • Three bacterial strains are differentiated simultaneously by labeling DNA with different reporter groups and visualizing the hybridization signal with reporter-specific detector proteins labeled with different fluorophores.
  • each bacteria in a mixture of all seven organisms can b identified.
  • the total assay time is as short as 1.5 hours.
  • a dental plaque sample from a patient with localized juvenile periodontitis, an oral mixed microbial infection reveals numerous hybridization positive bacteria when probed with DNA from A. actinomycetemcomitans, the putative microbial pathogen for this disorder.
  • This example demonstrates the usefulness of the in situ hybridization methods of the present invention for the identification of individual non-cross hybridizing bacteria in mixed cell populations.
  • the seven bacterial species analyzed above are facultative or obligate anaerobes that are often found in association with inflammatory periodontal disease.
  • the role of these microorganisms is uncertain due to the lack of rapid and reliable methods to identify specifies.
  • Direct analysis of oral microbial specimens circumvents biases imposed by culture methods and may allow a better understanding of the role of these bacteria in the pathogenisis of periodontal disease.
  • Data on the detection of A. actinomycetemcomitans in a specimen from a patient with localized juvenile periodontitis indicates that the above-described in situ hybridization procedure facilitates such studies.
  • swabs taken from early culture plates may be streaked on a slide and assayed by in situ hybridization using probes for suspected pathogens of interest.
  • the present invention permits one to increase further the number of simultaneously detectable bacteria without increasing the number of available fluorophores by using combinatorial fluorescence imaging (Nederiof, P.M. et al., Cytometry 77: 126-131 (1990)).
  • combinatorial fluorescence imaging Nederiof, P.M. et al., Cytometry 77: 126-131 (1990)
  • the resultant hybridized probe will be detected by more than one of the fluorescent detectors and the signal will appear in more than one of the separate flurorescence images.
  • fluorescence signals appearing at the same site on two images will be "blended" and generate a new pseudocolor that is distinguishable from either of the originals.
  • the above example demonstrates the feasibility of differentiating multiple bacterial species (such as those found in the periodontal microflora) using multiparametric fluorescence in situ hybridization (M-FISH) and digital imaging microscopy.
  • M-FISH multiparametric fluorescence in situ hybridization
  • digital imaging microscopy By monitoring both hybridization signal specificity and bacterial mo ⁇ hology, seven different bacteria are readily and simultaneously distinguished in a mixed population using only three distinct fluorophores.
  • Such methods are readily applicable to the detection of bacteria (as well as viruses, and/or lower eukaryotes) that may be present in other clinical samples (in eluding those that contain complex mixtures of microflora (e.g., stool, saliva, throat swabs, sputum, vaginal secretions, etc.) and those that are normally (e.g., central spinal fluid (meningitis), blood (sepsis), etc.) or non-clinical samples (food products, water reservoirs, ecosytems, etc.).
  • microflora e.g., stool, saliva, throat swabs, sputum, vaginal secretions, etc.
  • those that are normally e.g., central spinal fluid (meningitis), blood (sepsis), etc.
  • non-clinical samples food products, water reservoirs, ecosytems, etc.
  • the in situ hybridization and digital imaging fluorescence microscopy methods of the present invention can simultaneously differentiate seven microorganisms in a laboratory derived mixed sample.
  • This technique provides both single cell detection sensitivity and the ability to correlate hybridization signal specificity with microbial mo ⁇ hology.
  • the relative abundance of a specific organism in a complex mixture or bacteria can be readily assessed and a single hybridization positive bacteria can be detected in an excess of 10 other bacteria.
  • the assays can be completed in less than 1.5 hours.
  • none of the genomic DNAs from the seven bacteria used in the above study exhibited cross-hybridization. To illustrate the capacity of the present invention to differentiate among related
  • Capnocytophaga DF 1 strains C. gingivalis (DR2001), C. spumblea (D4), and C. ochracea (C25) Such strains exhibit cross-hybridization (Rubin, S.J., Eur. J. Clin. Microbiol 5:253-257 (1984)).
  • the strains are obtained from the NTH Institute of Dental Research, Bethesda, MD. Bacterial DNA is isolated as described above.
  • Probes are made as follows: total genomic bacterial DNAs is labeled by nick translation using biotin- 11-dUTP (BIO), digoxigenin-11-dUTP (DIG) or dinitrophenol-11-dUTP (DNP) as described by Lichter, P. et al. (Hum. Genet. 80:224-234 (1988)). Uninco ⁇ orated nucleotides are removed using a Sephadex G-50 spin column equilibrated with 10 mM Tris-HCl/ 1 mM EDTA/0.1% SDS, pH 8.0. Labeled DNAs (2-6 ⁇ g) are ethanol precipitated and redesolved in 100% deionized formamide.
  • Capnocytophaga encompasses a group of fusiform, gram-negative, capnophilic, fermatative, gliding bacteria (Ledbetter, E.R. et al, Arch. Microbiol. 722:17-27 (1979)) that are common inhabitants of the oropharyngeal flora (Holdeman, L.V. et al, J. Periodon. Res. 20:475-483 (1985)).
  • C. gingivalis, C ochracea and C. spumblea have been implicated in gingivitis in children (Moore, W.E.C. et al, Infect. Immun.
  • Capnocytophaga DF- 1 species shows the pathogenic potential of this genus and may reflect differences in clinical significance of the respective species in medically important infections and periodontal disease (von Graevenitz, A., Eur. J. Clin. Microbiol. 5:223-224 (1984)).
  • Conventional methods of identifying bacteria from oral sources requires first, isolation of single bacterial colonies from the oral microbial milieu, and second, the assignment of isolates to genus and species based upon cellular mo ⁇ hology, gram stain, fermentation of carbohydrates and hydrolysis of various substrates. These procedures require considerable time and expertise, taking upwards of 2 weeks to identify single species from clinical samples.
  • a major drawback of this method is that DNA from 10 3 to 10 6 microorganisms of interest are required for such assays, therefore bacteria in low abundance in a mixed population are likely not to be detected.
  • cross- hybridization which is common in closely related species, can impede identification to the species level.
  • the intensity of the hybridization signal (i.e.. fluorescence photon output) from a bacteria (“bacteria A”) A will be greatest with a genomic DNA derived from that bacteria.
  • Related strains which share some DNA sequences in common with a DNA probe of genomic DNA for bacteria A will give less intense hybridization signals, which directly reflect the extent of sequence homology.
  • Unrelated bacteria that share little or no sequence homology to bacteria A will yield virtually no fluorescence signal at all.
  • Computer thresholding of fluorescence images is used to establish the extent of cross-hybridization (i.e., sequence homology) between the three Cagnocytophaga species.
  • pure samples of each bacteria are separately hybridized with biotinylated DNA probes from all three species.
  • three slides are prepared that contain only C. ochracea cells. The first slide is hybridized in situ using BlO-labeled C. ochracea genomic DNA, the second slide is hybridized in situ using BlO-labeled C. gingivalis genomic DNA and the third slide is hybridized in situ using BlO-labeled C. spumble genomic DNA. The slides are then washed and hybridization positive bacteria are identified using FlTC-avidin.
  • C. ochracea and C. gingivalis did not crosshybridize with one another, however C. spumblea cross-hybridized with both C. gingivalis and C. ochracea.
  • a computer analysis of the intensity of the fluorescent hybridization signals indicates that C. spumblea had significant sequence homology with C. ochracea ( ⁇ 22%) but much less (- 5%) with C. gingivalis.
  • Hybridization is accomplished using 15 ⁇ l of hybridization solution which contained 50 ng of labeled probe and DNAse treated salmon sperm DNA (15 ⁇ g) in 50% (vol/vol) deionized formamide/2xSSC (0.3 M sodium chloride/0.03 M sodium citrate, pH 7.0)/ 5% dextran sulfate. The solution is applied to the sample, covered with a coverslip and sealed with rubber cement. Both bacterial DNA and labeled DNA probe are denatured by heating at 80°C for 5-8 minutes in an oven. These steps are sufficient to permeabilize the bacteria for probe assessability.
  • the DNAs are allowed to reassociate by incubating the slides at 37°C for 2.5 min. in a moist chamber. Posthybridization washings, blocking and detection are conducted as described by Lichter, P. et al (Hum. Genet. 80:224-234 (1988)). Briefly, the slides are washed 3 times in 50% formamide/ 2xSSC at 42°C for 5 minutes and then washed 3 times in O.lxSSC at 60°C for 5 minutes. The slides are then incubated in a solution of 3% bovine serum albumin/4xSSC (blocking solution) for 30 minutes at 37°C.
  • Biotinylated probes are detected using fluorescein isothiocyanate-(HTC)- avidin DCS (Vector Laboratories, 5 ⁇ g/ml), Cascade Blue-avidin (Molecular Probes Inc., 10 ⁇ g/ml) or Rhodamine-avidin (Boehringer Mannheim, 10 ⁇ g/ml).
  • Digoxigenin-labeled probes are detected using FTTC or Rhodamine conjugated sheep anti-digoxigenin Fab fragments (Boehringer Mannheim, 2 ⁇ g/ml). Dinitrophenol labeled probe is detected by incubating with rat anti-DNP antibodies (Novagen.
  • bacterial DNA was counterstained with 4,6-diamidino-2- phenylindole (DAPI) at a concentration of 200 ng/ml.
  • DAPI 4,6-diamidino-2- phenylindole
  • fluorochrome- conjugated antibodies or avidin are diluted into a solution of 4xSSC/l% BSA and 0.1% tween-20 (200 ⁇ l/slide) and incubated with the sample at 37°C for 30 minutes in the dark. The slides are then washed 3 times in 4xSSC/0.1% tween-20 at 42°C prior to viewing. Hybridization is visualized via epifluorescence microscopy using a Zeiss
  • the Gene Join Max Pix software can be used in a second way to achieve bacterial speciation with genomic DNA probes.
  • This embodiment exploits the fact that when separate fluorescence images (e.g., fluorescein and rhodamine) are merged to form a composite image; the source image with the greatest fluorescent intensity (i.e., photon output) at each pixel location will be color-dominant in the merged image.
  • This attribute permits the use of multiple differentially labeled genomic DNA probes simultaneously to probe a single or mixed bacterial population. Since each bacteria in a mixture will exhibit the greatest fluorescence signal with the genomic probe that is 100% homologous, the image merging process results in effective speciation.
  • ochracea probe and the Rhodamine fluorescence imge produced by the C. spumblea probe also show both intense signals for some bacteria and weak signals for others, reflecting interspecies hybridization.
  • each bacterium shows an intense signal in one of the three images and a weaker (or absent) signal in the other images (reflecting interspecies hybridization). Accordingly, through the use of thresholding (i.e., using the signal generated to determine which probe gave the more dominant signal with which bacteria), the method enables one to determine the species of each bacterium.
  • the in situ hybridization methods of the present invention provide a rapid and simple method for the speciation of cross-hybridizing microorganisms, such as the oral Capnocytophaga.
  • the basis of this method relies on the finding that all microorganisms within different genera and species have unique DNA sequences within their genome. Closely related organisms will have some DNA sequences in common, and the amount of such cross-hybridizing sequences will directly affect their ability to be differentiated by in situ hybridization.
  • the fluorescence images obtained from in situ hybridization with each detector which reflect the amount of hybridization of a bacterium to each of the genomic DNA probes, are digitalized via computer. If a bacteria has no cross-hybridizing sequence with any of the other bacteria it will hybridize only to its homologous DNA and thereby be detected by only a single detector and its presence will be found in only the image co ⁇ esponding to that detector. However, if a bacteria does have cross-hybridizing sequences with one of the other bacteria, then it will be detected by the DNA probes from each bacteria and its presence will be seen in the corresponding images for each probe (detectors).
  • each detector e.g., Rhodamine, FTTC or Cascade Blue
  • the intensity of the signal generated in each image will be directly proportional to the amount of hybridization of a bacterium with a DNA probe.
  • the corresponding images from each detector are pseudocolored and overlaid (merged) to form a composite image. Since any given pixel on the monitor can only be a single color, only the most intense signal at each pixel is shown, and displayed as the corresponding pseudocolor.
  • M-FISH multiparametric fluorescence in situ hybridization
  • C. gingivalis C ochracea
  • C. Sproda C.
  • Speciation of these three Capnocytophaga species can be achieved in as little as 30-90 minutes, markedly faster than the 2-3 weeks required using conventional microbiological methods.
  • the general discrimination strategy reported here is applicable to the broad spectrum of microbial pathogens for which an extent of sequence homology is known.
  • Nucleic acid amplification technology has greatly increased the ability to investigate detailed questions about the genotype or the transcriptional phenotype in small biological samples, and has provided the impetus for many significant advances in biology, especially in the field of genetics.
  • M-FISH multiparametric fluorescence in situ hybridization
  • a 6-fluor tagging approach permits the simultaneous identification of 63 different types of signals by virtue of their unique spectral signatures.
  • the M-FISH method of the present invention can distinguish and identify all of the chromosomes of a human karyotype.
  • the invention permits the determination of whether a particular karyotype contains the gene, allele or chromosomal element in question.
  • a probe specific for a deletion, rearrangement, insertion or other mutation characteristic of a genetic disease e.g., hemophilia, cystic fibrosis, breast or other cancer, etc.
  • it is possible to diagnose whether a patient exhibits the genetic disease e.g., to diagnose cystic fibrosis, etc.
  • the invention permits one to determine whether a patient's chromosomes carry a recessive allele associated with genetic disease.
  • the invention permits one to determine whether a patient is predisposed to a disease by virtue of the presence of a genetic lesion (e.g., a mutation in the apoE, p53, rb, or brcall2 genes) associated with a future disease state (such as Alzheimer's Disease, heart disease, cancer, etc.).
  • a genetic lesion e.g., a mutation in the apoE, p53, rb, or brcall2 genes
  • a future disease state such as Alzheimer's Disease, heart disease, cancer, etc.
  • the M-FISH method of the present invention can distinguish and identify the species of microorganisms present in a clinical or non-clinical sample.
  • the invention permits the determination of whether a particular microorganism contains the specific gene, allele or chromosomal element.
  • M-FISH a probe specific for an antibiotic resistance determinant, a cellular antigen, a toxin, etc.
  • the combined use of M-FISH and nucleic acid amplification permits a determination of whether a particular microbial or viral strain is resistant to an antibiotic, or is pathogenic, or expresses a toxin.
  • Such combination of methodologies thus permits the serotyping and subspeciation of pathogens without any requirement of culturing and/or purification.
  • the M-FISH methods of the present invention particularly concern probe sets and methods that are sufficient to characterize both the genotypic and phenotypic charateristics of micobes present in a preparation.
  • a genotypic probe is one capable of specifically hybridizing to phylogenetically related species of microbes; hybridization of such a probe to nucleic acid of a preparation thus reveals whether a preselected microbe, or its cross-hybridizing (i.e., phylogenetically) related microbes are present in such a preparation.
  • a phenotypic probe is one capable of specifically hybridizing to genes, or genetic elements associated with a particular phenotype (e.g., antibiotic resistance, toxin production, antigen presentation, etc.).
  • the invention particularly contemplates the multiparametric use of multiple fluors, and particularly concerns the embodiments in which 2, 3, 4, 5, 6 or more fluorophores are employed so as to permit the detection and/or characterization of 3, 7, 15, 31, 63 or more combinations of genotypic or phenotypic elements.
  • 2, 3, 4, 5, 6 or more fluorophores are employed so as to permit the detection and/or characterization of 3, 7, 15, 31, 63 or more combinations of genotypic or phenotypic elements.
  • 31 genotypic/phenotypic elements can be probed.
  • Any combination of such elements can be employed.
  • 25 of such elements can be genotypic elements (thus permitting the identification of 25 different species of microbes)
  • 6 of such elements can be genotypic (for example permitting the determination of whether any of the 25 identified species are resistant to any of 4 antibiotics or present any of 2 toxins).
  • 10 of such elements can be genotypic elements (thus permitting the identification of 10 different species of microbes), whereas 21 of such elements can be genotypic (for example permitting the determination of whether any of the 10 identified species are resistant to any of 9 antibiotics, present any of 3 toxins, present any of 4 surface antigens, and express any of 5 genes).
  • E. coli strains and strains of other enterics e.g., Salmonella
  • Clostridria Vibrio, Corynebacteria, Listeria, Bacilli (especially B. anthracis), Staphylococcus Streptococci (especially beta-hemolytic Streptococci and S. pneumoniae), Borrelia, Mycobacterium (especially M. tuberculosi); Neisseria (especially N. gonorrhoeae), Trepanoma.
  • enterics e.g., Salmonella
  • Clostridria Vibrio, Corynebacteria, Listeria, Bacilli (especially B. anthracis), Staphylococcus Streptococci (especially beta-hemolytic Streptococci and S. pneumoniae), Borrelia, Mycobacterium (especially M. tuberculosi); Neisseria (especially N. gonorrhoeae), Trepanoma.
  • bacteria implicated in periodontal disease e.g., Fusobacteria, Porphyromonas, Eikenella, Prevotella, Actinobacillus, and Capnocytophaga species), etc.
  • viruses e.g., parvoviruses, papoviruses, he ⁇ esviruses, togaviruses, retroviruses (especially HIV), rhabdoviruses, influenza viruses, etc.
  • lower eukaryotes fungi (e.g., Dermatophytes; Pneumocystis, Trypanosoma; etc.), yeast, helminths, nematodes, etc.) can be detected and characterized using such a method.
  • the combined use of a nucleic acid amplification method and the multiparametric fluorescence in situ hybridization methods of the present invention can be used to explore the quiescence or expression state of cells.
  • the methods of the present invention can determine not merely the presence of tumor cells, but the extent of their malignancy.
  • Such mRNA profiling may be conducted even in circumstances in which standard cDNA hybridization approaches may not be sensitive enough to detect changes in the concentration of low abundancy gene products.
  • a nucleic acid amplification method and the multiparametric fluorescence in situ hybridization methods of the present invention can employ any suitable amplification method or methods (e.g., Polymerase Chain Reaction (Mullis, K. et al, Cold Spring Harbor Symp. Quant. Biol. 57:263-273 (1986); Erlich H. et al, EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al, US 4,683,202; Erlich, H., US 4,582.788; Saiki, R. et al, US 4,683,194 and Higuchi, R. "PCR Technology," Ehrlich, H.
  • Rolling Circle Amplification is an amplification strategy in which nucleic acid amplification is driven by a DNA polymerase that can replicate circularized oligonucleotide probes with either linear or geometric kinetics, under isothermal conditions.
  • RCA involves incubating at least one one rolling circle replication primer (RCRP) with at least one amplification target circle (ATC).
  • the ATC comprises a single stranded circular DNA molecule that contains a region complementary to the RCRP, such that the RCRP can hybridize to the ATC and mediate amplification in the presence of a DNA polymerase.
  • the DNA product remains bound at the site of synthesis, where it may be tagged, condensed, and imaged as a point light source.
  • Linear oligonucleotide probes bound covalently on glass surfaces can generate RCA signals, whose color indicates the allele status of the target, depending on the outcome of specific, target-directed ligation events.
  • Single molecule counting by RCA provides a powerful mutation detection method for studies using oligonucleotide arrays, and is particularly amenable for the analysis of rare somatic mutations.
  • the combined M-FISH / nucleic acid amplification methods of the invention may be used to detect circularizable oligonucleotides, called "padlock probes" (Landegren, U. et al, Methods 9:84-90 (1996); Landegren, U. et al, Ann Med. 29:585-590 (1997); (Nilsson, M. et al, Science 265:2085-2088 (1994); Nilsson, M. et al, Nat. Genet. 16: 252-255 (1997), all herein inco ⁇ orated by reference) bound to single copy genes in cytological preparations.
  • padlock probes Landegren, U. et al, Methods 9:84-90 (1996); Landegren, U. et al, Ann Med. 29:585-590 (1997); (Nilsson, M. et al, Science 265:2085-2088 (1994); Nilsson, M. et al, Nat. Genet. 16: 252-255
  • any DNA sequence thus captured into a circular DNA may be amplified by RCA or HRCA.
  • the reaction may thus be advantageously used in situations where it is desirable to interrogate the sequence inco ⁇ orated into a padlock probe at some point after RCA.
  • Longer sequences, such as microsatellite repeats should also be capable of being copied into circularizable probes for amplification and analysis. Subsequent to such copying, since there is little likelihood that rolling circle amplification will modify the number of repeats inco ⁇ orated into a circularized probe (Fire, A. et al, Proc. Natl. Acad. Sci. (USA) 92:4641-4645 (1995)), direct measurement of repeat size would be feasible.
  • any of three alternative HRCA/RCA strategies for allele discrimination utilizing ligation of circularizable DNA probes and rolling circle replication may be employed in concert with the M-FISH methods of the present invention.
  • the polymerase mediated "gap-fill” reaction is preferred over the "gap probe” ligation reaction for solution studies of complex genomes.
  • the gap oligonucleotides which cannot be washed away in a solution assay, are preferably used at relatively high concentrations for the ligation step, and may therefore interfere with the HRCA reaction, inducing the formation of amplicon artefacts.
  • the gap ligatior reaction is considered preferable for allele analysis of DNA in cytological specimens.
  • the specificity of ligation should be enhanced relative to probes without a gap because three different sequence recognition events and two independent ligation events must occur before "padlock" closure.
  • the third assay method ligase-mediated extension of an oligonucleotide linked to a solid surface, provides a totally novel approach to quantify individual hybridization/ligation events and to score rare somatic mutations.
  • the DNA generated by RCA is labeled with combinatorially labeled fluorescent DNP-oligonucleotide tags that hybridize at multiple sites in the tandem DNA sequence.
  • the "decorated" DNA, labeled by specific encoding tags, is then condensed into a small object by cross- linking with a multivalent antibody (e.g., anti-DNP IgM).
  • the wild-type specific primer generates RCA products which can hybridize to fluorescein-labeled DNP- oligonucleotide tags, while the mutant RCA products hybridize to Cy3-labeled DNP- oligonucleotides.
  • CACHET Condensation of Amplified Circles after Hybridization of Encoding Tags.
  • the present invention provides an ability to simultaneously identify the twenty-four different human chromosomes in a metaphase spread by hybridizing a complete set of chromosome-specific DNA probes, each labeled with a different combination of dyes.
  • One aspect of the present invention is the recognition that, through the use of telomere-specific probes, the methods of the present invention may be used to identify translocations that would be non-identifiable (i.e., cryptic) through the use of conventional methods.
  • the present invention provides, for the first time, a simple screening test to assess the integrity of telomeric regions using a single hybridization reaction.
  • Such multiplex hybridization assays significantly improve the ability to detect terminal deletions and cryptic chromosomal rearrangements, and thereby facilitate the identification of structural abnormalities that elude detection with conventional cytogenetic banding or multicolor whole - chromosome painting methods.
  • this aspect of the present invention extends prior efforts in both karyotyping technology (see, e.g., Speicher, M.R. et al, Nature Genet 72:368-375 (1996); Speicher, M.R. et al, Bioimaging 4:52-64 (1996); Schrock, E.
  • telomere integrity assay technology see, e.g., Ledbetter, D.H., Amer. J. Hum. Genet. 57:451-456 (1992)); all herein inco ⁇ orated by reference).
  • This probe set employed a microdissected probe for the short arm of the Y chromosome (Guan, X.Y., Nature Genet. 72: 10-11 (1996), herein inco ⁇ orated by reference) because no suitable sub-telomeric YAC for the short arm could be identified.
  • the probe set did not include probes for the p arms of the acrocentric chromosomes 13, 14, 15, 21 and 22.
  • the probes are combinatorially labeled such that the telomeric regions of these chromosomes are visualized in different pseudocolors based upon their unique fluorophore composition.
  • the p arm and q arm telomere proximal probes for each chromosome are labeled with the same "color code,” for example both chromosome 1 telomeres are labeled in color "a,” while the chromosome 2 telomeres are labeled in color "b,” etc.
  • any cytogenetically cryptic chromosome translocation occurring on a non- acrocentric chromosome are visualized in the metaphase spreads as a chromosome harboring 2 colors rather than a single color per chromosome.
  • the 24 color telomere integrity assays utilize a complex probe mixture, such probe cocktails can be hybridized with high reproducibility.
  • Such hybridization is visualized, as described above, with, for example, an epifluorescence microscope equipped with an appropriate filter set and a cooled CCD-camera, etc.
  • the fluors and high contrast filters are the same as those described above.
  • telomeric probe set may be composed of a mixture of YACs, Half-Yacs
  • telomere sequences of the molecule are provided by human DNA rather than yeast DNA.
  • Half- YACs contain sub- telomere repeats that are known to reside on different chromosomes thus, in some instances, they hybridize to multiple telomeres.
  • specific M-FISH signals can be obtained from half- YACs, for example through the use of DNA amplification (using, for example, PCR with Alu primers to reduce the relative representation of repeat sequences); addition of Cot-1 DNA to the hybridization cocktail will suppress hybridization of any residual repetitive sequences.
  • Suitable YACS, half- YACs and/or CePH-YACs are described by Vocero-Akhani, A. et al.
  • CEPH-YACs were selected to contain the most telomeric genetic markers. Because the distance from the true telomeric end of the chromosome is unknown, these probes were tested by FISH for their distal band location. For six telomeric regions (2p, 2q, 5q, 7q, 8p, 8q) the hybridization of a single sub-telomeric YAC yielded low fluorescence intensities. Therefore two (5q, 7q, 8p, 8q) or three (2p, 2q) subtelomeric YACs were pooled in order to increase the signal. Alu-f ⁇ nge ⁇ rinting analysis was used to confirm that the YACs overlap.
  • the terminal regions of the chromosomes are specifically analyzed. Chromosome ends are identified and segmentation masks with different thresholds are calculated and checked for regions with increased fluorescence intensities. This is necessary because the simultaneous hybridization of multiple small region-specific probes results in signals that are often in different focal planes. In the absence of Z-axis optical sectioning and image merging, this results in some YAC probes giving significantly reduced fluorescence intensity values. If a specific telomeric region is not labeled with a particular fluor, no increased fluorescence intensity peaks is observed in at least one of the segmentation masks.
  • the calculated fluor segmentation masks were not overlayed with the DAPI segmentation mask that delineates the chromosomal boundaries (since some of the telomeric FISH signals can lie outside the DAPI segment mask).
  • Individual YAC-clones first are assigned distinct gray values depending on the boolean signature of each probe or the combination of fluors used to label it (Speicher, M.R. et al, Bioimaging 4:52-64 (1996); Schrock, E. et al, Science 273:494-491 (1996)). A look-up-table then is used to assign each DNA target a psuedocolor depending on this gray value. Finally, this pseudocolored image was overlayed onto the DAPI-stained chromosome image that was assigned a light blue color.
  • the multiplex telomere probe set is hybridized to normal metaphase spreads from peripheral blood lymphocytes. Similar to the results described above obtained with whole chromosome painting probes, YAC probes that are labeled with equal amounts of different fluors do not always give equal signal intensities for each fluor. To diminish signal intensity differentials, probe concentrations for the hybridization mix and a reliable combinatorial labeling scheme are therefore established by control experiments. YACs yielding large fluorescence signals are preferentially labeled with three different fluors while YACs yielding rather weak signals were labeled with one fluor only.
  • telomere integrity assay is conducted on karyotypes of 10 normal male and female donors. No indication of polymo ⁇ hisms are detected thereby suggesting that cryptic translocations are extremely infrequent in normal populations. A typical metaphase spread is observed ( Figure 5A), and a normal karyotype based on the boolean signature of our subtelomere probes is attained as expected ( Figure 5B).
  • the probe set is hybridized on metaphase spreads from a patient with a myeloproliferative disorder.
  • Karyotyping using G-bands reveals trisomy 8 as the only detectable cytogenetic change. This is verified by M-FISH using chromosome specific painting probes.
  • the telomere integrity assay yields an intriguing hybridization pattern on the chromosomes 8.
  • the trisomy 8 is verified in all 10 metaphase spreads analyzed. However, on 8 of these 10 metaphase spreads a split telomere signal is observed on two chromosomes 8, indicating an inversion in this region ( Figure 6). This analysis demonstrates the power of this new technique to detect structural abnormalities that are undetectable by standard cytogenetic methods or 24-color techniques using chromosome specific painting probes.
  • telomere-integrity assay may be very rewarding. evaluating karyotypes of patients with mental retardation, a combination of mental retardation and dysmo ⁇ hic features, and cancer cells that are known to have a high genomic instability. The latter cells should be highly predisposed to sub-telomeric translocation events. Screening for cryptic translocations can not be done efficiently with chromosome specific painting probes because they were not designed to detect subtle deletion or rearrangment events.
  • the telomere-specific probe sets can be improved using PCR-assisted chromatography (Craig, J. et al, Hum. Genet 100:412-416 (1997), herein inco ⁇ orated by reference).
  • This tool removes repetitive DNA, including the polymo ⁇ hic repetitive sequences from half-YACs or other M-FISH probes, and thereby facilitates the generation of probes that will hybridize specifically in the absence of Cot-1 suppressor DNA.

Abstract

Cette invention a trait à un ensemble de sondes oligonucléotidiques marquées de manière combinatoire dont chaque élément, (i), est porteur d'une marque prédéterminée pouvant être distinguée de celle de tout autre élément dudit ensemble et, (ii), est capable d'hybridation spécifique avec un chromosome ou une molécule d'acide nucléique prédéterminés. Elle porte également sur l'utilisation de ces molécules, seules ou associées à des techniques d'amplification d'acide nucléique.
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